Role of an Active Site Loop in the Promiscuous Activities of <i>Amycolatopsis</i> sp. T‑1-60 NSAR/OSBS

The <i>o</i>-succinylbenzoate synthase (OSBS) family is part of the functionally diverse enolase superfamily. Many proteins in one branch of the OSBS family catalyze both OSBS and <i>N</i>-succinylamino acid racemization in the same active site. In some promiscuous NSAR/OSBS enzymes, NSAR activity is biologically significant in addition to or instead of OSBS activity. Identifying important residues for each reaction could provide insight into how proteins evolve new functions. We have made a series of mutations in <i>Amycolatopsis</i> sp. T-1-60 NSAR/OSBS in an active site loop, referred to as the 20s loop. This loop affects substrate specificity in many members of the enolase superfamily but is poorly conserved within the OSBS family. Deletion of this loop decreased OSBS and NSAR catalytic efficiency by 4500-fold and 25,000-fold, respectively, showing that it is essential. Most point mutations had small effects, changing the efficiency of both NSAR and OSBS activities <10-fold compared to that of the wild type. An exception was F19A, which reduced <i>k</i>_cat/<i>K</i>_M^OSBS 200-fold and <i>k</i>_cat/<i>K</i>_M^NSAR 120-fold. Mutating the surface residue R20E, which can form a salt bridge to help close the 20s loop over the active site, had a more modest effect, decreasing <i>k</i>_cat/<i>K</i>_M of OSBS and NSAR reactions 32- and 8-fold, respectively. Several mutations increased <i>K</i>_M of the NSAR reaction more than that of the OSBS reaction. Thus, both activities require the 20s loop, but differences in how mutations affect OSBS and NSAR activities suggest that some substitutions in this loop made a small contribution to the evolution of NSAR activity, although additional mutations were probably required

Comparison of <i>Alicyclobacillus acidocaldarius</i> <i>o</i>‑Succinylbenzoate Synthase to Its Promiscuous <i>N</i>‑Succinylamino Acid Racemase/<i>o</i>‑Succinylbenzoate Synthase Relatives

Studying the evolution of catalytically promiscuous enzymes like those from the <i>N</i>-succinylamino acid racemase/<i>o</i>-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, <i>Alicyclobacillus acidocaldarius</i> OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds <i>N</i>-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 10² M^–1 s^–1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects <i>k</i>_cat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily